1. Field of the Invention
This invention relates to the field of polypeptides having antimicrobial activity and the polynucleotides encoding them. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid constructs. The invention more specifically relates to an antimicrobial fusion polypeptide comprising a truncated ply187 endolysin from the Staphylococcus aureus phage 187. The invention further relates to compositions and a method of making the polypeptides and method of treating staphylococcal-associated diseases, including methicillin-resistant Staphylococcus aureus (MRSA).
2. Description of the Relevant Art
Staphylococcus aureus is a pathogen that causes a broad spectrum of human and animal diseases and has adapted to antibiotic selective pressures resulting in a high prevalence of multi-drug resistant strains (de Lencastre et al. 2007. Curr. Opin. Microbiol. 10:428-435). The spread of these antibiotic-resistant strains is a threat to public health and a critical concern to health care providers worldwide.
Phage endolysins are cell wall hydrolases that are produced near the end of the phage lytic cycle to help the nascent phage escape the infected host. Endolysins are ideally suited as antimicrobials for several reasons as described previously (Loessner, M. J. 2005. Curr. Opin. Microbiol. 8:480-487; Donovan et al. 2009. Biotech. International 21:6-10). Most importantly, phage endolysins are believed to have co-evolved with their host such that they target cell wall bonds that are believed essential and difficult for the host cell to alter. Thus bacterial resistance is unlikely. Due to the absence of an outer membrane in Gram-positive bacteria, endolysins are able to kill these bacteria when added from without.
The S. aureus bacteriophage 187 endolysin (Ply187) gene was initially reported by Loessner et al. (1999. J. Bacteriol. 181:4452-4460). Ply187 consists of 628 amino acids and has a calculated molecular mass of 71.6 kDa. Typically, endolysins from a Gram-positive background have a modular structure with an N-terminal catalytic domain for peptidoglycan hydrolysis and a C-terminal cell wall binding domain (Loessner, supra). However, the Pfam domain database indicates that the amino terminus of Ply187 harbors a Cysteine, Histidine-dependent Amidohydrolase/Peptidase (CHAP) domain (Bateman and Rawlings. 2003. Trends Biochem. Sci. 28:234-237; Rigden et al. 2003. Trends Biochem. Sci. 28:230-234) and the C-terminus contains a glucosaminidase domain with no known C-terminal cell wall binding domain (Loessner et al., supra; FIG. 1A). Cell wall binding domains are essential for the lytic activity of some endolysins and often determine specificity (Baba and Schneewind. 1996. EMBO J. 15:4789-4797; Grundling and Schneewind. 2006. J. Bacteriol. 188:2463-2472; Loessner et al. 2002. Mol. Microbiol. 44:225-349; Lu et al. 2006. J. Biol. Chem. 281:549-558; Sass and Bierbaum. 2007. Appl. Environ. Microbiol. 73:347-352).
The Phage K endolysin, LysK, has been shown to kill a wide range of staphylococci including multiple MRSA in plate lysis assays (O'Flaherty et al. 2005. J. Bacteriol. 187:7161-7164). Blast analysis of the LysK protein sequence reveals two lytic domains, a CHAP endopeptidase domain, an amidase (N-acetyl-muramyl-L-alanine amidase) domain, and a C-terminal SH3b cell wall binding domain (O'Flaherty et al., supra). It is common for phage endolysins to have an N-terminal lytic domain (or two) with a C-terminal cell wall binding domain (Loessner, M. J. 2005, supra), although recently an endolysin with two lytic domains flanking two mid-protein cell wall binding domains (Cpl-7) was reported for the LambdaSa2 prophage (Pritchard et al. 2007. Appl. Environ. Microbiol. 73: 7150-7154).
Novel antimicrobials that are specific for staphylococcal species, including methicillin-resistant Staphylococcus aureus (MRSA) and that are also refractory to resistance development are needed to contend with the rise of drug-resistant pathogenic bacteria.